JP2012135300A - Microbial strain and cultivation method which show yield increase and inhibitory effect on late blight disease in solanaceous plant and which show protective effect on yield decrease due to continuous cropping in leguminous plant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microbial strain, which makes low cost cultivation possible and also has a great effect of both promoting growth and inhibiting late blight disease, concerning cultivation techniques in the seedling stage and the early growing stage after transplanting of the solanaceous plants, particularly of the genus Capsicum, and also to provide a microbial stain which has an effect of inhibiting yield decrease of leguminous plants, particularly of the genus Glycine, in the continuously cropped field.SOLUTION: The microbial strain is Arthrobacter oxydans, accession number FERM P-22038, and the cultivation method is to inoculate the seeds of solanaceous or leguminous plants with a microbial strain before growing the seeds.

Description

  The present invention relates to a microbial strain that can be suitably used in the cultivation of eggplants such as peppers and peppers and legumes such as soybeans, and a cultivation method using the same.

  In many of the production areas of Kyoto traditional vegetables such as Fushimi Kancho Pepper and Manganji Pepper, semi-forcing or forcing cultivation is carried out in which seedlings grown in pots are planted in farmhouse fields. Such a cultivation method can increase the profitability by advancing the shipping time from the usual, but by raising the seedlings over three months from December or January, the growth of seedlings due to low temperature frequently occurs, Moreover, a great deal of labor is required. Moreover, since the planting time is also the early spring low temperature period (February to March), the initial growth for 1 to 2 months after planting is slow, the cultivation period is long, and the production efficiency is poor. In addition, soil diseases are likely to occur during this period. When a plague occurs, farmers are severely damaged because many individuals die and become unable to harvest. Therefore, there is a demand for technological development for growing healthy seedlings that are resistant to soil diseases.

  As a technique for promoting the growth of vegetables and the like using microorganisms, VA mycorrhizal fungi, which are plant symbiotic fungi, are commercially available and distributed as a government-designated soil improvement material by the Geopower Promotion Act (Non-patent Document 1). Except for fungi, there are many examples of research on growth promotion technology using a group of microorganisms called plant growth promoting rhizosphere bacteria overseas, but there are few cases in Japan (Non-patent Document 2). On the other hand, many researches have been conducted on biological control technology that attempts to control diseases using microorganisms (Non-patent Document 3). Currently, efforts are being made to register agricultural chemicals mainly by agricultural chemical-related companies. Yes.

  Furthermore, unlike microorganisms that exhibit any one of the growth promoting effects and disease control effects as described above, as the only material that fulfills both purposes as long as the inventors have investigated, plant growth promoting rhizobacteria and chitin, etc. The composition (patent document 1) containing an organic soil improvement agent is proposed.

Special table 2003-529539

List of Ordinance-designated Soil Improvement Materials (Characteristics / Application Methods / Handling Companies), [online], [Search May 31, 2010], Internet <http://www.japan-soil.net/dokai-shizai/2_shizai .htm> Development of plant growth-promoting rhizosphere bacteria utilization method in spinach, [online], [Search May 31, 2010], Internet <http://www.cgk.affrc.go.jp/seika/seika_print/materials/ report_06 / report_2007_06f.pdf> Pest control technology using biological pesticides, [online], [October 19, 2010 search], Internet <http://www.pref.saga.lg.jp/web/library/at-contents/shigoto/ nogyo / nougyougijutsu / jizoku / pdf / A7.pdf>

However, using each of the microorganisms showing the growth promotion effect and the microorganisms showing the disease control effect increases the cost significantly for the farmer. In addition, the composition described in Patent Document 1 has an unclear effect on plants other than the plants identified in the examples because the range of target plants is too wide, and there is no description of the treatment method for plants. It is clear.
Therefore, the first object of the present invention is to solve the above-mentioned problems relating to the cultivation technique of the solanaceous plant, particularly Capsicum, at the time of raising seedlings and at the initial growth after planting, and is highly effective for both growth promotion and prevention of epidemics. It is in providing the strain which also has. A second problem is to provide a strain having an effect of preventing a decrease in yield in a continuous cropping field of legumes, particularly a soybean genus.

The strain of the present invention that solves the problem is
It is a microbial strain of Arthrobacter genus oxydans species accession number FERM P-22038 that has the property of promoting the growth of solanaceous plants and suppressing the occurrence of plague, and also the property of preventing the decrease in yield due to continuous cropping of legumes .
When the solanaceous plant belongs to the genus Capsicum, or when the legume plant belongs to the genus Soybean, the effects of the present invention are particularly remarkable.
The invention related to the invention of the strain is a cultivation method characterized by inoculating the microorganism strain on a seed of a solanaceous plant or legume and growing the seed.

  The strain of this invention has the property of promoting the growth of solanaceous plants and suppressing the occurrence of plague. Therefore, when the strain of this invention is used for cultivation of solanaceous plants, growth can be promoted and the occurrence of epidemics can be suppressed, and the farmer can cultivate stably and efficiently at low cost. In addition, the strain of the present invention has a property of preventing a decrease in yield in a continuous cropping field of legumes. Therefore, when the strain of the present invention is used for cultivation of legumes, the same field can be used continuously for many years.

Example 1
(1) Isolation of rhizosphere bacteria A vigorous strain of about one month after planting was collected from local fields of pepper, eggplant, and leek maintaining high productivity, and a separation source was prepared by an underwater fractionation method. Next, it was cultured at 30 ° C. for 24 to 48 hours by a dilution plate method using King B agar medium. From the colonies formed after culture, colonies that fluoresce under UV irradiation are picked, and as shown in Table 1, 188, 196, 200 strains from the pepper, eggplant and leek strains, respectively, a total of 584 isolates Got. This is because it has been empirically known that fluorescent bacteria are excellent in affinity with plants and highly likely to have a positive effect on the growth of plants.
These isolates were suspended in a skim milk dispersion medium, stored frozen at −80 ° C., and subjected to the following assay.

(2) Primary selection based on growth-promoting effect of isolated strains on red pepper (nursery seedling test)
Since there are many isolated strains, it is necessary to efficiently select a strain having a growth promoting effect among them. Therefore, the following procedure was used to grow capsicum inoculated with the isolated strain in a growth chamber in the laboratory, and selection was performed at the seedling stage immediately after germination.

  Dissolve skim milk dispersion medium frozen with the isolated strain at room temperature, dilute with water, inoculate on a flat medium, select typical colonies of the colonies that appear after growth, and select Becton, Ditzkinson and Using a liquid culture medium Trypticase soy broth made by a company, the cells were cultured with shaking at 30 ° C. for 24 hours. Then, the cells collected by precipitation with a centrifuge were suspended in sterilized water to prepare about 109 cfu / ml. The strain was inoculated into the pepper seed by immersing the pepper seed (variety “Fushimi Amaga Togarashi”, Takii Seed Co., Ltd.) in this suspension for 1 hour.

  Separately, vermiculite was put into a vial (made of glass, inner diameter 35 mm × height 75 mm), about 1/3 of the bottle capacity, and 10 ml of a commercially available liquid fertilizer Hyponex (registered trademark) 2000 times solution was added thereto as a nutrient. And the said pepper seed | inoculated with the isolate was seed | inoculated to the vermiculite in a bottle. After growing for 14 days in a growth chamber (25 ° C., 16 hours light / 8 hours), the foliage weight and root weight were measured. In determining the inoculation effect, a non-inoculation zone was established as a control and compared with this.

  As a result of the seedling test in the growth chamber, strains with the same leaf weight as compared to the control but with a significantly increased root weight (growth promotion type), and strains with the same root weight but a significantly reduced leaf weight (Growth suppression type) was observed. Among these, 6 strains that showed a relatively high growth promoting effect were used as primary selection strains (Table 2) and subjected to secondary selection by a pot test.

(3) Secondary selection based on growth-promoting effect on red pepper of isolated strain (pot test)
In order to examine the growth promotion effect of the primary selected strains in more detail, the inoculated pepper was subjected to a pot test in the following procedure in an environment (in a glass greenhouse) close to normal cultivation (nurturing) conditions.
The primary selected strain was inoculated into the pepper seeds in the same manner as in the primary selection. After seeding capsicum seeds inoculated with the strain in a vinyl pot (φ7.5 cm) filled with soil (trade name “Prosoil”, manufactured by Shinto Chemical Co., Ltd.) and growing in a glass greenhouse for 1 month, The fresh weight of the foliage and roots was measured. The growth promoting effect was determined by comparing the measured value with the non-inoculated control group.
Many of the primary selection strains exceeded the control group in both stem and leaf weight and root weight, but the growth promoting effect on the root of KP9670 strain was remarkable (Table 3).

(4) Examination of epidemic pathogenesis control effect of isolated strain In order to obtain a strain having both growth promotion effect and epidemic pathogenesis control effect, the potash epidemic pathogenesis control effect of the strain obtained by the primary selection was tested by pot test as follows. .
The isolated strain was cultured with shaking at 30 ° C. for 24 hours using the trypticase soy broth as in the first selection. Inoculate the roots of the seedlings by immersing the roots of 3 weeks old seedlings of the same kind of red pepper in the sterilized water suspension (about 109 cfu / ml) of the obtained cells for 1 hour. did.

  Separately, after cultivating Pepper pesticidal fungus (Phytophthora capsici strain P-04) in wheat bran / vermiculite medium for 2 weeks, the medium was crushed with a homogenizer, and the soil (trade name “Prosoil”, Shinto Chemical Co., Ltd.) The soil was prepared by mixing at a rate of 0.5% (w / v). Then, this diseased soil was filled into a vinyl pot (φ7.5 cm).

Next, the capsicum seedlings inoculated with the strain are planted in the plague soil in the vinyl pot, the disease incidence (wilt strain rate) is investigated over time, and the area under the pathological progression curve for 2 weeks after planting the disease ( area under disease progress curve (AUDPC) was compared with an uninoculated control group to evaluate the disease-causing effect.
As a result, as shown in Table 4, among the strains obtained by the primary selection, the KP9660 strain had the smallest AUDPC value, that is, the highest disease suppression effect. From these, it was considered that this strain is promising as a strain having both the effect of promoting the growth of pepper seedlings and the effect of suppressing the onset of the plague.

(4) Effect of inoculation of KP9660 strain on the yield of red pepper We examined the effect of increasing the yield of KP9660 strain selected as a strain having both the growth promotion effect of pepper seedling and the control effect of epidemic disease in the same manner as in the first selection Capsicum seeds were inoculated. After seeding inoculated capsicum seeds of KP9660 strain in a vinyl pot (φ7.5 cm) filled with soil (trade name “Prosoil”, manufactured by Shinto Chemical Co., Ltd.) and growing in a glass greenhouse for 1 month Planted in a farm field (gray lowland), fertilized and managed in accordance with the practices of Kyoto Prefecture. 28 days after planting, harvesting started, harvested every 7 to 10 days for 40 days, the number of fruits harvested per individual and the weight of fruit per individual were measured, and compared with the uninoculated control group To determine the effect of increasing sales.

  As a result, the KP9660 inoculated group exceeded the uninoculated control group in terms of the number of harvested fruits and the weight of the fruit, and a high yield increase effect was confirmed. The excellent product rate was also higher than the control. The excellent product rate was determined as the ratio of the weight of the excellent fruit among all the harvests, defining a straight fruit with a length of about 15 cm and no bending as an excellent product.

(5) Identification of KP9660 strain The bacterial species was identified from the morphological characteristics, biochemical properties, and 16S rRNA gene base sequence of the KP9660 strain.
This strain is a gram-staining-positive short bacillus, but its cell shape is not constant, such as showing a V shape in the early stage of growth. Spores do not form and grow aerobically. It does not utilize glucose, is catalase positive, does not show motility, and does not grow at 4 ° C and 41 ° C. Oxidase positive, gelatin liquefaction negative, arginine dehydrogenase negative, denitrification negative, starch degradation positive, levan production negative were exhibited, and weak fluorescent pigment production was observed when cultured on King B agar medium. In addition, as a result of examining the partial base sequence (described below) of the 16S rRNA gene and performing a BLAST search, it showed 99.7% homology with the base sequence of the known Arthrobacter oxydans DSM201119 strain.
From the above results, the KP9660 strain was identified as Arthrobacter oxydans Sguros. The KP9660 strain has been deposited at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology under the deposit number FERM P-22038.

-Example 2-
(1) Cultivation of KP9660 strain KP9660 strain was dissolved in potato-peptone liquid medium (dissolved the following components in 1000 ml of potato 200 g: peptone 5.0 g, disodium phosphate · 12 hydrate 3.0 g, phosphate 1 The mixture was cultured with shaking at 28 ° C. for 24 hours in 0.5 g of potassium, 3.0 g of sodium chloride, pH 7.0). Centrifugation was performed at 10,000 G for 20 minutes while cooling at 5 ° C., and the supernatant (medium) was removed, and then the precipitate (cells) was suspended in the same amount of sterile water as the medium. Centrifugation and suspension of the cells in sterilized water were repeated twice to remove the medium components, that is, the cells were washed, and finally the cells were suspended in the same amount of sterile water as the medium. The bacterial concentration of this suspension was 1.0 × 10 9 cfu / ml, and this was used as the inoculation source.

(2) Effect of inoculation of KP9660 strain at the cell seedling raising stage of soybean Filling 72 hole cell tray with Napra soil (S type, made by Yanmar) and seeding soybean (variety: Shintanba black) one by one in one hole did. Immediately after the covering, 2 ml of the inoculum, that is, the cell suspension of KP9670 strain was poured from the top of the covering. Then, it was grown to a stage where it could be planted in a farm by growing for 10 days while pouring water in a greenhouse. Then, when the foliage weight, root weight, foliage length and germination rate were measured, the seedlings inoculated with the same strain had a higher germination rate than the control (no inoculation), the foliage length was low and the root weight was heavy, The seedlings were healthy seedlings (Table 6). The inoculation method was not only the method of pouring the cell suspension after sowing, but also the same effect was obtained not only by soaking the seeds in the suspension for a short time and then sowing.

(3) Effect of inoculation of KP9660 strain in a continuous cropping field of soybean For a field where remarkable yield reduction occurs due to continuous cropping, two different soil types (gray lowland and brown lowland) were tested, and the method of (2) Soybean cell seedlings inoculated and grown with the KP9660 strain were planted at a planting density of 2000 strain / 10a in late June 2010. In the test field, cattle manure bark compost is 1t / 10a in early May 2010, ammonium sulfate (N content 20%) is used as the basic fertilizer and 1kg / 10a is used in late May 2010. In the middle of the month, 2 kg / 10a (each nitrogen component amount) was applied. Other cultivation management was in accordance with Kyoto Prefecture customary standards (Kyoto Bean Cultivation Guide, Kyoto Prefectural Agriculture, Forestry and Fisheries Department, 2006). The gray lowland soil field evaluated the inoculation effect by grain yield (crude grain weight), and the brown lowland soil field by green soybean yield (salmon weight).

  As a result, in the gray lowland / continuous cropping field, the grain yield in the control plot (no inoculation) was 21.7 kg / strain, but in the KP9660 inoculation plot, it increased to 46.3 kg / strain, more than doubled. (Table 7). The grain yield in the control plot is 43.3 kg in terms of 10a, and it can be judged that this is a very low-yielding field, about half of the average yield of about 100 kg. At such a yield level, effective measures are not found, and cultivation is usually abandoned. Therefore, the effect of increasing the yield by inoculating this strain is significant. In brown lowland soil / continuous cropping field, the yield of green soybeans in the control group (no inoculation) was 103.6 g / strain, but in the inoculation group of KP9660 strain, it increased to about 126.9 g / strain in the inoculation group (Table). 8). The yield of green soybeans in the control plot is 207.2 kg in terms of 10a, which is a very low-yielding field that is about half the prefectural average of about 400 kg. It was confirmed that the inoculation effect of this strain was observed even in the case of low yielding soybean harvest.

(4) Effect of inoculation of KP9660 strain in non-continuous cropping field of soybean Inoculation method and cultivation method are the same as in (3), and a gray lowland soil field where the soybean is not continuously cropped The inoculation effect of this strain was confirmed. As a result, the grain yield in the control group (no inoculation) increased to 90.8 kg / strain in the KP9660 inoculation group compared to 75.0 kg / strain (Table 9). The grain yield in the control plot was 150.0 kg in terms of 10a, which can be judged as a field with higher productivity than the prefecture average yield of about 100 kg. Therefore, it was confirmed that the inoculation effect was exhibited even in the field where the yield did not decrease.

Claims (4)

  A microbial strain of Arthrobacter genus oxydans species accession number FERM P-22038, which has the property of promoting the growth of solanaceous plants and suppressing the occurrence of plague, and also preventing the decrease in yield due to continuous cropping of legumes.   The microbial strain according to claim 1, wherein the solanaceous plant belongs to the genus Capsicum.   The microbial strain according to claim 1, wherein the leguminous plant belongs to the genus Soybean.   A cultivation method comprising inoculating a seed of a solanaceous plant or legume plant with the microbial strain of claim 1 and growing the seed.